Phytochemical Analysis

Phytochem. Anal. 17: 25–31 (2006)

GLYCYRRHIZIN AND GLYCYRRHETIC ACID IN LIQUORICE Phytochemical
25

Published online 28 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pca.877

Analysis

Simultaneous HPLC Analysis, with Isocratic Elution, of

Glycyrrhizin and Glycyrrhetic Acid in Liquorice Roots
and Confectionery Products
CESARE SABBIONI,1 ANNA FERRANTI,1 FRANCESCA BUGAMELLI,1 GIORGIO CANTELLI FORTI,2 AND
MARIA AUGUSTA RAGGI1*
1
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
2
Department of Pharmacology, Faculty of Pharmacy, Alma Mater Studiorum, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy

Received 15 May 2004; Revised 17 February 2005; Accepted 17 February 2005

Glycyrrhizin (1), the main active principle of Glycyrrhiza glabra (liquorice) roots, is extensively used in herbal medicines, in phar-
maceutical preparations and confectionery products. A feasible and reliable method which allows the simultaneous analysis of 1
and its aglycone, 18β -glycyrrhetic acid (2), by means of an isocratic HPLC procedure is described. The system uses a C8 column as
the stationary phase, and a mixture of acetonitrile, methanol, water and glacial acetic acid as the mobile phase. Good linearity was
found in the concentration ranges 1–50 and 0.05–2.50 µg/mL for 1 and 2, respectively. A simple and rapid sample pre-treatment,
based on the extraction of the two analytes with a mixture of water and ethanol, was developed for the examination of liquorice
confectionery products and root samples. The HPLC method was shown to be appropriate, in terms of precision and feasibility, for
the quality control of the analytes in these matrices. Copyright © 2005 John Wiley & Sons, Ltd.

Keywords: Liquid chromatography; glycyrrhizin; glycyrrhetic acid; confectionery products; liquorice root; Glycyrrhiza glabra.

INTRODUCTION

Glycyrrhiza glabra (liquorice) roots and rhizomes are

extensively used in herbal medicines for their emollient,
anti-tussive, anti-inflammatory, anti-viral and gastro-
protective properties.
The product referred to as ‘liquorice’ in the manu-
facture of confectionery (i.e. chips or cylinders of
pure liquorice) is obtained by treating dried roots of
G. glabra with boiling water, which is then evaporated
to obtain a semi-solid extract that may be subjected
to further treatment to obtain different commercial
products. Liquorice extract is also used as a masking
agent or taste corrective in several pharmaceutical
formulations (e.g. in preparations containing cascara,
ammonium chloride and quinine) and in food produc-
tion (e.g. to improve the taste of beer).
The main active compound of G. glabra is
glycyrrhizin {1; glycyrrhizic acid, [3-O-(2-O-β -
D -glucopyranuronosyl- α - D -glucopyranuronosyl)-3 β -
hydroxy-11-oxo-18β, 20β -olean-12-en-29-oic acid]}, a
saponin with a pentacyclic triterpenic structure bound
to two glucuronic acid molecules. The content of 1
(as potassium and calcium salt) in liquorice roots is
between 2 and 15% by weight of the drug depending on
the species and the geographic and climatic conditions

* Correspondence to: M. A. Raggi, Department of Pharmaceutical

Sciences, Faculty of Pharmacy, Alma Mater Studiorum, University of
Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
Email: mariaaugusta.raggi@unibo.it

Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 17: 25–31 (2006)
DOI: 10.1002.pca
26 C. SABBIONI ET AL.
(Fenwick et al., 1990; Spinks and Fenwick, 1990). The
most readily appreciable characteristic of 1 is its sweet
taste, from which it derives its name ‘Glycyrrhiza’
which means ‘sweet root’ in ancient Greek. In fact, 1 is
170 times sweeter than sucrose (Mizutani et al., 1994),
and this is the basis of its use in the food industry and
in pharmaceutical products.
Owing to its known pharmacological properties as an
anti-tumour (Ukiya et al., 2002), anti-viral (Baltina,
2003) and anti-ulcer agent (Doll et al., 1962), much
research interest has been shown in the aglycone of
1, 18β -glycyrrhetic acid (2), that is readily formed by
hydrolysis or metabolism of 1 and may also be present
in very small amounts in the plant. Glycyrrhizin is
one of the leading natural compounds to be taken
to clinical trials with regard to chronic active viral
hepatitis and human immunodeficiency virus (HIV)
infections. It has been suggested that the demon-
strated anti-viral activity of 2 is due to its antioxidant
properties (Baltina, 2003; Fujioka et al., 2003; Liu
et al., 2003). Studies have shown that long-term treat-
ment of 1 prevents the development of hepatic carci-
noma from C hepatitis (Van Rossum et al., 1998), and
recently it has been found that 1 has in vitro anti-viral
activity against SARS-associated coronavirus (Cinatl
et al., 2003). Compound 1 may also be co-administered
with pharmaceutical formulations containing oestrogen
derivatives to inhibit unwanted effects such as
alterations in blood coagulation and thrombosis
(Francischetti et al., 1997); on the other hand, a ‘simil-
oestrogenic’ activity of 2 has been demonstrated and
its use in the substitutive treatment of menopausal
dysfunctions has been proposed (Sharaf et al., 1975).
Recent clinical and pharmacological studies have
verified the high level of safety of 1. In fact, side effects
such as cardiac dysfunctions, oedema, weight gain and
hypertension have only been found in predisposed sub-
jects or in those receiving very high doses of pure 1.
These effects, however, were shown to be less frequent
and less severe in subjects receiving liquorice extract
containing the same amount of 1 (Bernardi et al.,
1994). Pharmacological studies on both humans and
rats have reported a significant decrease in the
bioavailability of 1 when it is administered as liquorice
extract, as opposed to the pure compound (Cantelli
Forti et al., 1994a).
From a consideration of the above, there is clearly a
need for a simple analytical method by which to deter-
mine 1 and 2 in plant roots, pharmaceutical formula-
tions, confectionery products and plasma samples.
Several papers report analytical methods for the deter-
mination of 1 and 2 in different matrices including
commercial products and biological fluids (Hurst et al.,
1983; Ichikawa et al., 1984; Collinge et al., 1985;
Hermesse et al., 1986; De Groot et al., 1988; Spinks
and Fenwick, 1990; Yamamura et al., 1991; Raggi
et al., 1994a; Okamura et al., 2001). Such procedures
Copyright © 2005 John Wiley & Sons, Ltd.
are mostly based on reversed phase HPLC with UV
(Hurst et al., 1983; De Groot et al., 1988; Raggi et al.,
1994b; Collinge et al., 1985; Okamura et al., 2001) or
fluorometric detection (Yamamura et al., 1991). Most of
these methods analyse 1 and 2 separately, and only
two of them simultaneously determine both analytes
using HPLC with gradient elution (Okamura et al.,
2001) or column switching (De Groot et al., 1988).
To the best of our knowledge, no method has been
reported that permits the determination of both 1 and
2 by isocratic liquid chromatography. The aim of this
paper was to develop a rapid, facile and economic
HPLC method for the simultaneous analysis of 1 and 2
in confectionery products and in roots of G. glabra.
EXPERIMENTAL
Reagents and samples
Methanol, acetonitrile (HPLC-grade), glacial acetic
acid and 96% ethanol (pure for analysis) were from
Carlo Erba (Milan, Italy). Ultrapure water (18.2 MΩ cm)
was obtained using a Millipore (Milford, MA, USA)
MilliQ apparatus. Glycyrrhizin ammonium salt (ca.
75% pure), glycyrrhetic acid (ca. 95% pure) and
indomethacine (3; internal standard) were purchased
from Sigma (St. Louis, MO, USA). Stock solutions of 1,
2 and 3 were prepared at concentrations of 1 mg/mL
in methanol, and were stable for at least 5 months
when stored at −20°C. Working solutions were pre-
pared every day by diluting the stock solutions with the
HPLC mobile phase.
The different liquorice-based confectionery products
analysed were purchased from stores in Bologna (Italy).
Pure extracts, ‘Oronero’ chips and cylinders (Sirea,
Reggio Emilia, Italy) and ‘Saila liquirizia purissima
extraforte’ (Saila, Silvi Marina, TE, Italy) contained
pure liquorice extract and natural flavours. ‘Tabù’
candies (Perfetti S.p.A., Lainate, MI, Italy) contained
pure liquorice flavoured with mint. Roots of Glycyrrhiza
glabra were of commercial quality from Saila and Sirea.
Chromatographic protocols
Isocratic system. The HPLC apparatus consisted of
an Agilent (Waldbronn, Germany) 1100 series
isocratic pump and an Agilent 1100 series photodiode
array detector (PAD). The data system consisted of
an Hewlett Packard CORE Chemstation LC 3D
(Waldbronn, Germany). Isocratic separation was
achieved using an Agilent Zorbax Eclipse XDB C8
reversed-phase column (150 × 4.6 mm i.d.; 5 µm) and
a Phenomenex (Torrance, CA, USA) SecurityGuard
C8 pre-column (4.0 × 3.0 mm i.d.; 5 µm). The mobile
phase, consisting of methanol: acetonitrile:water:glacial
Phytochem. Anal. 17: 25–31 (2006)
DOI: 10.1002.pca

Table 1 Composition of the mobile phase employed in the
gradient HPLC system
Time Compositiona of
(min) mobile phase (%)
A B
0 99
10 95
11 90
13 80
14 75
15 65
17 60
18 50
45 50
a (55 mm diameter; Whatman, Maidstone, UK) and an
Solvent A, acetonitrile:water:glacial acetic acid (35:64:1,
v/v/v); solvent B, methanol.
acetic acid (35:35:30:1, by volume), was filtered through
Millipore nylon filters (47 mm diameter; 0.2 µm pore
size) and degassed by sonication (Transsonic T-310
instrument; Elma, Singen, Germany) prior to use.
Separations were carried out at room temperature
with a flow rate of 1 mL/min and an injection loop of
20 µL. The PAD detector was operated in the range
200–320 nm, and quantitative analysis was performed
at 254 nm.
Gradient system. For the purposes of validation, a
gradient system was also employed to analyse the
same samples. The HPLC apparatus consisted of a
Jasco (Tokyo, Japan) PU-1580 gradient pump and
a Jasco UV-970 spectrophotometric detector set at
254 nm. Separations were achieved on an Agilent
Zorbax Eclipse XDB C8 reversed-phase column (150 ×
4.6 mm i.d.; 5 µm) and a Phenomenex SecurityGuard
C8 pre-column (4.0 × 3.0 mm i.d.; 5 µm). The mobile
phase was composed of acetonitrile:water:glacial acetic
acid (35:64:1, by volume; solvent A) and methanol
(solvent B). Components of the mobile phase were
filtered through Millipore nylon filters (47 mm
diameter; 0.2 µm pore size) and degassed by sonication
prior to use. The composition of the mobile phase was
varied during the run according to the nonlinear
gradient scheme shown in Table 1. Separations were
carried out at room temperature with a flow rate of
1 mL/min and an injection loop of 20 µL. After the end
of each chromatographic run, the system was allowed
to equilibrate under initial conditions for 30 min.
Method validation
Calibration curves. Standard solutions in the
concentration range 1–50 µg/mL for 1 and 0.05–
Copyright © 2005 John Wiley & Sons, Ltd.
GLYCYRRHIZIN AND GLYCYRRHETIC ACID IN LIQUORICE 27
20 µg/mL for 2, were prepared and injected into the
HPLC system: the internal standard 3 was maintained
at a concentration of 1 µg/mL. The analyte peak
area values were plotted against the corresponding
concentrations of the analytes (expressed in µg/mL)
and the calibration curves constructed by means of the
least-squares method.
1
5 Extraction procedure. The confectionery products or
10 roots were ground to a fine powder and a sample
20 (200 mg) transferred to a 50 mL round bottom flask
25 together with 1 mL of the standard solution of 3 and
35 19 mL of ethanol:water (1:1, v/v). The mixture was
40 maintained by thermostat at 60°C for 25 min with
50 stirring and then centrifuged for 10 min at 3000 rpm.
50
The supernatant was filtered through a paper filter
aliquot of the filtrate, suitably diluted with mobile
phase, was subjected to HPLC analysis.
Precision. In order to evaluate intermediate precision
(inter-day precision) and repeatability (intra-day
precision), assays were performed by extracting and
injecting the same sample six times on the same day
and six times over different days. Each assay was
carried out at three different concentrations of 1 (1, 10
and 20 µg/mL) and of 2 (0.05, 0.10 and 2.50 µg/mL).
The percentage relative standard deviations (RSD%) of
the data thus obtained were calculated.
Accuracy. Accuracy was evaluated by means of
recovery assays carried out by adding standard
solutions of the analytes to the samples. The amounts
of analytes added corresponded to concentrations of 1,
10 and 20 µg/mL for 1 and 0.05, 0.10 and 2.50 µg/mL
for 2. The mean recoveries of the added analytes were
then calculated.
RESULTS AND DISCUSSION
HPLC method
From our previous experience concerning the analysis
of glycyrrhizin (1) and glycyrrhetic acid (2) in different
matrices (Raggi et al., 1994a, b), it was known that
an acid mixture of acetonitrile and water and an acid
mixture of methanol and water worked well as a mobile
phase for the chromatographic analysis of 1 and 2,
respectively; however, none of these mixtures were
suitable for the simultaneous determination of both
analytes. For this reason, a mobile phase containing
different ratios of acetonitrile, methanol, water and
glacial acetic acid was employed. Table 2 shows the
retention times of the analytes obtained with different
compositions of mobile phase. The results show that
it is necessary to balance the amounts of the organic
Phytochem. Anal. 17: 25–31 (2006)
DOI: 10.1002.pca
28 C. SABBIONI ET AL.
Table 2 Chromatographic behaviour of glycyrrhizin (1) and glycyrrhetic acid (2) with
isocratic elution with different HPLC mobile phases
Composition of mobile phase (%)
Water Methanol Acetonitrile
64.5 15 20
54 16.5 29
50 25 25
29 35 35
Figure 1 HPLC chromatogram of a standard solution of
glycyrrhizin (1) and glycyrrhetic acid (2) (10 µg/mL) and
indomethacine (3) (1 µg/mL) obtained following isocratic
elution with a mobile phase containing acetonitrile:
methanol:water:glacial acetic acid (35:35:29:1, by volume) at a
flow rate of 1 mL/min. (For chromatographic protocol see the
Experimental section.)
solvents in order to obtain good retention times for
both 1 and 2, which are two chemically very different
substances, the former being highly hydrophilic (due
to the two glucuronic acid groups), whilst the latter is
lipophilic. A mixture of acetonitrile, methanol, water
and acetic acid (35:35:29:1) was found to be optimal
for this purpose. Quantitative analyses were carried at
a wavelength of 254 nm, where both analytes showed
a relative absorbance maximum, and PAD analysis
allowed unambiguous peak identification (by means
of on-line spectra) and analysis of peak purity.
Indomethacine was selected as internal standard and
was also used for retention time control. Figure 1
shows the chromatogram of a standard solution con-
taining 1, 2 (10 µg/mL of each) and 3 (1 µg/mL); all
peaks are symmetrical and well separated with reten-
tion times of 3.0 min for 1, 4.5 min for 3, and 16.0 min
for 2.
Different linearity ranges were studied for the two
analytes, according to the amount of each substance
expected to be present in the matrix, that is, 1 is
usually >4% in liquorice root and 3–5% in ethanolic
extract (European Pharmacopoeia, 2005), whilst 2 is
typically present only in trace amounts. Good linearity
Copyright © 2005 John Wiley & Sons, Ltd.
Retention time (min)
Acetic acid Glycyrrhizin Glycyrrhetic acid
0.5 25 >60
0.5 10 >60
1 5.1 33.6
1 2.9 16
was obtained with standard solutions over the concen-
tration range 1–50 µg/mL for 1 and 0.05–2.50 µg/mL
for 2. The limit of detection (LOD) and limit of
quantitation (LOQ) were calculated according to recom-
mendations of the United States Pharmacopeia (2005)
and were, respectively, 4 and 10 ng/mL for 1 and 8
and 20 ng/mL for 2. The precision of the chromato-
graphic method was evaluated and acceptable RSD%
values were obtained: the repeatability data were
within the ranges 0.6–1.4% for 1 and 0.6–1.8% for 2,
and the intermediate precision data varied from 1.2 to
1.6% for 1 and from 1.0 to 2.1% for 2. The full set of
validation parameters is reported in Table 3.
Extraction procedure
The chemical characteristic of the analytes should be
carefully considered when developing an extraction
procedure for 1 and 2 owing to their rather different
chemical–physical properties. Compound 1 is very
soluble in water, whereas 2 is almost insoluble in
water but soluble in alcohol. Different solvents, previ-
ously employed by other authors, were tested for the
extraction of 1 and 2 from confectionery and liquorice
roots including water: ethanol (80:20; Lauren et al.,
2001), 2 M ammonium hydroxide (Frattini et al., 1977;
Okamura et al., 2001), and water (Ong and Len, 2003).
The amounts of analytes obtained when extracting the
same sample with the three solutions are reported in
Table 4. Extraction with water (carried out for the
industrial production of liquorice sweets) gave the
lowest recovery, especially for 2. Extraction with 2 M
ammonia was tested to exploit the acidic properties of
the analytes; however, the extraction yields were not
satisfactory. It was found that a mixture of water and
ethanol was the best extraction medium but required
continuous infusion with hot ethanol:water (20:80,
v/v) for 4 h. In order to reduce the length of the extrac-
tion procedure, the percentage of ethanol was in-
creased to 50%, obtaining a quantitative extraction in
25 min. Thus, this latter procedure was chosen for
subsequent assays.
In order to confirm that the extraction procedure with
this medium was quantitative, a sample of confectionery
Phytochem. Anal. 17: 25–31 (2006)
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 GLYCYRRHIZIN AND GLYCYRRHETIC ACID IN LIQUORICE 29

Table 3 Validation parameters for the developed isocratic HPLC method

Parameter Glycyrrhizin Glycyrrhetic acid

Linearity range (µg/mL) 1–50 0.05–2.5

Regression equationa y = 0.20445x + 0.04414 y = 0.57554x − 0.01823
Limit of detection
Standard solutions (µg/mL) 4 × 10−3 8 × 10−3
Confectionery products (%)b 0.002 0.004
Limit of quantitation
Standard solutions (µg/mL) 10 × 10−3 20 × 10−3
Confectionery products (%)b 0.005 0.010
Analyte concentration (µg/mL) 1 10.0 25.0 0.05 1 2.5
Repeatability (RSD%)c 1.4 0.8 0.6 1.8 1.4 0.6
Intermediate precision (RSD%)c 1.5 1.4 1.2 2.1 1.9 1.0

a
y = peak area; x = concentration (µg/mL).
b
Percentage in confectionery products corresponding to the reported LOD and LOQ values.
c
n = 6.

Table 4 Comparison of the efficiency of extraction of

glycyrrhizin (1) and glycyrrhetic acid (2) from the same sample
using different extraction solvents

Extraction solvent

Analyte extracted Water:ethanol Ammonium Water

(amount, µg/mL) (80:20, v/v) hydroxide (2 M)

Glycyrrhizin 11.7 10.6 9.3

Glycyrrhetic acid 0.27 0.25 0.17

product and a sample of root were extracted twice

with the extraction medium, and the solutions thus
obtained were injected separately into the HPLC. No
detectable analyte peak was found in the solutions
obtained from the second extraction step.
Figure 2 HPLC chromatogram of a sample of Sirea Oronero
chips following extraction with 20 mL of a mixture of water:
ethanol (1:1) and dilution (1:50) with the mobile phase. (For
a key to peak identity see the legend to Fig. 1; for chromato-
graphic protocol see the Experimental section).

Application to commercial confectionery products

and liquorice roots
Following validation of the extraction procedure
and the chromatographic method, six different com-
mercially available liquorice products were analysed.
The chromatogram of a confectionery product (Sirea
Oronero chips) after the extraction procedure and dilu-
tion with mobile phase (50 times), is shown in Fig. 2.
No interference with the resolution of 1, 2 or 3 was
observed, and the retention times remained the same
as for the respective standard materials. The analyte
concentrations in the sample, obtained by interpolation
on the appropriate calibration curves, were found to be
10.6 µg/mL of 1 and 260 ng/mL of 2. The amounts
of analytes found in the examined products are re-
ported in Table 5. The amounts of 1 were very similar
for the four different confectionery materials, and were
between 4.5 and 5.4% w/w, while 2 was present in
only trace levels. Moreover, the amount of 1 in roots
was lower than in the confectionery materials.
The precision of the method (extraction and chro-
matography) was then evaluated: intermediate preci-
sion values were obtained by repeating the extraction
of the same sample (Sirea Oronero chips containing
5.3% of 1 and 0.13% of 2) six times over different days;
the RSD% thus calculated was 3.0% for 1 and 3.5%
for 2. Accuracy was assessed using powdered commer-
cial product (Sirea Oronero chips) spiked with three
different levels of each analyte. Figure 3 shows the
chromatogram of the sample to which 10 µg/mL of 1
and 2 µg/mL of 2 had been added. The recovery values
were between 95 and 102% for 1 and between 99 and
108% for 2, thus indicating the satisfactory accuracy of
the method.

Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 17: 25–31 (2006)
DOI: 10.1002.pca
30 C. SABBIONI ET AL.
Table 5 Amounts of glycyrrhizin (1) and glycyrrhetic acid (2) in commercial liquorice
confectionery samples and roots
Amount Sirea Sirea
found (%)a Oronero Oronero
chips cylinders
Glycyrrhizin 5.31 4.50
Glycyrrhetic acid 0.13 0.04
a
Expressed on a w/w basis.
b
n.d. = not detectable (<LOD).
Figure 3 HPLC chromatogram of the same sample depicted
in Fig. 2 spiked with 10 µg/mL of glycyrrhizin (1) and 2 µg/
mL of glycyrrhetic acid (2). (For a key to peak identity see the
legend to Fig. 1; for chromatographic protocol see the Experi-
mental section).
Comparison of the developed isocratic method with
a gradient method
While isocratic elution is less expensive and less time-
consuming than gradient elution, co-elution with other
components of the matrix can occur, thus lowering the
selectivity of the method. For this reason, the same
liquorice extracts and confectionery products analysed
by the proposed method were analysed using a gradi-
ent method. The representative chromatograms of a
standard solution containing 1 and 2, and a liquorice
root sample are reported in Fig. 4(a and b), respec-
tively. As can be seen, the gradient method shows
greater resolution than the isocratic method but the
quantitative results derived from the gradient and
the isocratic method were in very good agreement.
For this reason, it can be concluded that none of the
compounds separated with the former method inter-
fered with 1 or 2 when employing the latter method.
Furthermore, the peak purities of 1 and 2 in confec-
tionery materials and root samples, as evaluated by
the Chemstation software, were always well within the
prescribed limits with both methods.
Copyright © 2005 John Wiley & Sons, Ltd.
Tabu Saila Saila Sirea
cylinders cylinders roots roots
5.37 4.91 2.96 4.03
n.d.b 0.03 0.25 n.d.
Figure 4 HPLC chromatograms obtained using a gradient
elution system (for chromatographic protocol see Experi-
mental section) of: (a) a standard solution containing
10 µg/mL each of glycyrrhizin (1) and glycyrrhetic acid (2),
and (b) a liquorice root sample.
The proposed HPLC method with UV detection,
coupled to a simple and rapid extraction procedure,
seems to be suitable, in terms of selectivity and sensi-
tivity, for the analysis of 1 and 2 in liquorice roots
and confectionery products. The method also has the
advantage of allowing for the simultaneous analysis of
1 and 2 with a single isocratic system. Compared with
other methods presented in the literature that employ
gradient systems or column switching (Ichikawa et al.,
1984; De Groot et al., 1988; Okamura et al., 2001), the
proposed method is simpler and requires less expen-
sive instrumentation. The sensitivity compared with
the method of De Groot et al. (1988) is significantly
higher and allows the quantivation of traces of 2 in
liquorice roots. Moreover, the extraction procedure
developed for roots and confectionery products allows
for the quantitative extraction of 1 and 2 in a single
step. The described HPLC method may also be applic-
able for the simultaneous determination of 1 and 2
levels in plasma for toxicological and pharmacokinetic
studies: further studies are in progress in order to
adapt the method to this type of biological matrix.
Phytochem. Anal. 17: 25–31 (2006)
DOI: 10.1002.pca

Acknowledgements
The authors wish to thank Dr. Simone Galassi for his
technical assistance.
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DOI: 10.1002.pca